Biocliem. J. (1993) 293, 859-866 (Printed in Great Britain) Oxygen Michaelis constants for tyrosinase Jose Neptuno RODRiGUEZ-LOPEZ,* Jose Ram6n ROS,t Ram6n VARON* and Francisco GARCiA-CANOVAStt *Departamento de Qu(mica-Fisica, E. U. Politecnica de Albacete, Universidad de Castilla-La Mancha, Albacete, and fDepartamento de Bioquimica y Biologia Molecular, Facultad de Biologia, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain The Michaelis constant of tyrosinase for oxygen in the presence of monophenols and o-diphenols, which generate a cyclizable o- quinone, has been studied. This constant depends on the nature of the monophenol and o-diphenol and is always lower in the presence of the former than of the latter. From the mechanism proposed for tyrosinase and from its kinetic analysis [Rodriguez- Lopez, J. N., Tudela, J., Varon, R., Garcia-Carmona, F. and INTRODUCTION Tyrosinase is a copper-containing enzyme that catalyses the ortho-hydroxylation of monophenols (monophenolase reaction) and the oxidation of o-diphenols to o-quinones (diphenolase reaction) [1]. The active site of tyrosinase consists of two copper atoms and three states, 'met', 'deoxy', and 'oxy' [2-5]. The Michaelis constant for oxygen of tyrosinase in the presence of o-diphenols has been widely studied. Ingraham determined the Km for 02 of French-prune tyrosinase in the presence of three o- diphenols [6]. In that study, a variation in the oxygen Michaelis constant according to the structure of the hydrogen donor was observed, and it was concluded that the enzyme combines with 02 before it does so with the o-diphenol [6]. The same variation was observed by Duckworth and Coleman [7], but they did not agree with Ingraham [6] and proposed that the reaction might follow the opposite order, oxygen not binding first. These authors proposed two binding sites [7]. The kinetic analysis of Neurospora crassa tyrosinase showed random binding of o-diphenol and 02 [8]. The complexity of the monophenol hydroxylation mechanism by tyrosinase has hindered the study of the oxygen Michaelis constant of this enzyme in the presence of monophenols, and the first mention found in the literature is by Lerch and Ettlinger [9]. They observed that the Km and kcat for the oxidation of L-3,4- dihydroxyphenylalanine (dopa) were strongly dependent on oxygen concentration, whereas those for L-tyrosine methyl ester were not [9]. These observations suggest that the enzyme was saturated by 02 acting on monophenols and not on o-diphenols, indicating that the Km, 2was lower in the presence of the former than of the latter, although no mechanism for the action of this enzyme was proposed. Lerner and Mayer [10] attempted to study the kinetic constant for oxygen in the monophenolase reaction, but did not succeed, concluding that the results indicated that the mechanism differed from that for o-diphenol. From the mechanism proposed for the monophenolase activity of tyrosinase [1 1] and its kinetic analysis [12], it was demonstrated that the o-diphenol accumulation in the steady state ([D],,,,) was linear with regard to monophenol concentration (MIo), and so [D]88 = R[T]0, where R is constant for each enzyme and each Garcia-Cainovas, F. (1992) J. Biol. Chem. 267, 3801-3810] a quantitative ratio has been established between the Michaelis constants for oxygen in the presence of monophenols and their o-diphenols. This ratio is used for the determination of the Michaelis constant for oxygen with monophenols when its value cannot be calculated experimentally. monophenol/diphenol pair [12]. These results permitted the development of a method for measuring the monophenolase activity of tyrosinase. This consists of adding to the reaction medium the o-diphenol necessary ([D],,8) to obtain the steady state. This method permits short assay times, with no lag period, no significant consumption of substrates, no suicide inactivation and no product breakdown. Therefore kinetic assays of the monophenolase activity can be carried out. The purpose of the present paper is to report a kinetic study that permits us to obtain the Michaelis constants for oxygen with both monophenols and o-diphenols in the tyrosinase-catalysed reaction. The value of Km for °2 in the presence of monophenols is given for the first time in the literature, and the validity of the mechanism proposed for tyrosinase is confirmed. NOTATION AND DEFINITIONS Species T D QH, Q L DC [XI [X]S8 Emet Edeoxy Eoxy monophenol o-diphenol o-quinone-H+ and o-quinone respectively leukoaminechrome aminechrome concentration of the species X during the course of the reaction concentration of the species X during the steady state of the reaction initial concentration of the species X in the assay medium met-tyrosinase or oxidized form of tyrosinase with Cu2+ in the active site deoxytyrosinase or reduced form of tyrosinase with CuI+ in the active site oxytyrosinase or oxidized form of tyrosinase with peroxide Kinetic parameters VT VD DC' DC steady-state rate of the production of DC from T and D respectively Abbreviations used: tyrosine, L-tyrosine; dopa, L-3,4-dihydroxyphenylalanine; dopachrome, 2-carboxy-2,3-dihydroindole-5,6-quinone; tyramine, 4- hydroxyphenethylamine; dopamine, 3,4-dihydroxyphenethylamine; dopaminechrome, 2,3-dihydroindole-5,6-quinone. : To whom correspondence should be addressed. Biochem. J. 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